Among physiologically active substances or environmental pollutants such as natural products, toxins, hormones or agricultural chemicals, numerous substances act in ultratrace amounts. Accordingly, instrumental analytical methods capable of performing high-sensitivity analysis have conventionally been widely used for qualitative and quantitative measurement of these substances. However, instrumental analytical methods are poor in specificity, require excessive time for analysis including pretreatment of samples, and comprise complicated operations. Thus, instrumental analytical methods are inconvenient for the purpose of rapid and convenient measurements that have been required in recent years. Meanwhile, immunoassays are highly specific and much easier in terms of operation than instrumental analytical methods. Therefore, immunoassays have gradually spread in the field of measurement of physiologically active substances and environmental pollutants. However, conventional immunoassays such as enzyme immunoassays using 96-well plates and latex agglutination assays do not always provide satisfactory rapidness and convenience for measurement or detection sensitivity.
Another need expected to be enabled is as follows. Achievement of higher sensitivity of tests that currently use relatively invasive samples such as swabs and blood makes it possible to detect very small amounts of analytes contained in relatively low-invasive samples such as snot, gargle water, and urine. Thus, it can be anticipated to develop a test method which has less burden on patients.
In recent years, a test kit (hereinafter referred to as an immunochromatography kit), in which an immunochromatography method (hereinafter also referred to as an immunochromatography or an immunochromato method) is used, has been used more often in examination of infections that require particularly rapid diagnosis. According to the spread of these kits, patients with infections can be identified by a rapid and convenient method, and subsequent diagnosis and therapy can be conducted immediately and accurately. For example, in an immunochromatography method using the sandwich method, a labeled second antibody capable of specifically binding to an analytical target (for example, an antigen) and a sample solution which may possibly contain the analytical target are spread on an insoluble thin-membrane support (for example, a glass fiber membrane, a nylon membrane, cellulose membrane, etc.) on which a first antibody capable of specifically binding to the analytical target has been immobilized in a specific region. As a result, an immune complex with the analytical target is formed in the region of the insoluble thin-membrane support on which the first antibody has been immobilized. The analytical target can be measured by detecting a signal such as the color development or coloring of a labeling substance. The labeling substance to be used herein may be, for example, a protein including an enzyme, colored latex particles, metal colloids, or carbon particles.
The immunochromatography method requires neither massive facilities nor instruments for determination and measurement. Furthermore, the immunochromatography method is simple in operation, and thus, measurement results are obtained only by adding dropwise a sample solution which may possibly contain an analytical target and then leaving it at rest for approximately 5 to 15 minutes. Thus, since the immunochromatography method promptly gives measurement results, this technique is widely used as a convenient, rapid, and highly specific method for determination and measurement in many scenarios, such as for clinical examination in hospitals and in assays in laboratories.
With regards physiologically active substances or environmental pollutants such as natural products, toxins, hormone or agricultural chemicals, and further samples in an initial phase of being infected with virus infection, many substances exert their effects in ultratrace amounts that are undetectable by conventional common immunochromatography methods. Therefore, there are demands for development of rapid, convenient, and highly sensitive immunochromatography methods for such substances.
On the other hand, by applying the principle of such an immunochromatography method, using a reader for optically measuring the optical density of a detection site, the presence of a test substance can be qualitatively confirmed, or such a test substance can also be quantified. As with the Cobas h232 series of Roche Diagnostics, there may also be a case in which the blood concentration of a protein known as a myocardial marker is promptly measured for approximately 15 minutes by quantifying it according to the immunochromatography method.
However, conventionally, the measurable range of the conventional immunochromato method using a gold colloid, a colored latex or the like as a labeling substance has been limited with regard to the low concentration range of a test substance due to the limitation of the absorbance per gold colloid particle or colored latex particle. Moreover, in order to solve the aforementioned problem, if the signal of gold colloid is amplified with silver ions, for example, the problem regarding low concentration range can be solved. However, the following problems would often occur (JP Patent Publication (Kokai) No. 2009-098139). That is, even if the amount of silver is increased, shield area is not changed so much, and thus, the absorbance in a high concentration range gets saturated. Further, if a large amount of gold colloid is used in order to ensure a measurable range in a low concentration range, a problem regarding false-positive results would be created by a non-specifically adsorbed gold colloid labeling substance, for example, together with the amplification of the signal. Thus, if the amount of the labeling substance is reduced to a level at which false-positive results are not obtained, there may be a case in which a phenomenon whereby an antigen becomes negative in an ultrahigh concentration range is observed due to what is called a prozone phenomenon.
The achievement of high sensitivity according to an enzyme method or a silver amplification method has been previously known (Japanese Patent No. 3309977; JP Patent Publication (Kokai) No. 2002-202307). However, there have been no systems capable of ensuring a wide sufficient measurable range from a low concentration range to a high concentration range.